What will happen if you inject free fatty acids?

[sdegrace]

Just to satisfy my curiousity. I've only recently started analysing FAME mixes (also some ethyl esters, sometimes derivatized to FAMEs, sometimes not), and I've just taken it for granted that you esterify the FFA before you analyze it. It just occurred to me to wonder why. What would happen if you had a mix of FFAs and didn't bother to derivatize it, just injected it on the GC with the same method using say the Restek FAMEWAX or Supelco Omegawax column?

I don't happen to have any free fatty acids on hand to look for myself, so I thought I would just ask, and maybe gain some insight into the rationale behind the way we do things.

Stephen

[chromatographer1]

Most of them will stay in the injection liner and slowly decompose to tar, water, and carbon dioxide.

Some may go onto your column and slowly migrate down the column unless they find an active site or a place where chemistry can take place.

There are always a few acid molecules that remain free acids or become anhydrides and are injected onto a column. They can damage the phase and or degrade the separations of your components.

best wishes,

Rod

[Bruce Hamilton]

Actually, it depends a little on how much you inject and how inert your injector system and column are.

The FFA usually appear as relatively-early eluting triangles when present in poorly-derivatised samples. The majority will, as Rod notes, accumulate in the inlet and increase activity, producing poor peak shapes and high baseline noise. They can crud up your column, which is expensive.

If the inlet system is inert ( or you use direct on-column injection), it's possible to analyse the FFA using a "FFA-rated" phase, but such phases are very fragile and can die quickly, and it's very, very difficlut to be quantitative, hence the use of derivatives.

Rod,

In case I don't catch you, all the very best for your well-earned retirement, and thank you very much for all the time and effort you have spent responding to enquires here. We all learn something new.

Remember, you can always resurrect yourself as a consultant if you get bored, but many people find retirement can be a very busy time, probably because there's one-less excuse to not do something...

I hope you find time to return here and chat, and always remember...

Please keep having fun,

Bruce Hamilton

[Consumer Products Guy]

FFA "as is" oftentimes produce ugly peak shapes; the industry (such as us) generally make into methyl esters for ease of chromatography. We use BF3-CH3OH or H2SO4-CH3OH to esterify FFA and soaps directly; triglycerides we saponify first, then esterify.

[Victor]

I am never sure what fatty acid means... certainly C12 and above acids are fatty acids, but are C2-C6 carboxylic acids "fatty acids"? I guess they are not....

There is no sense in trying to analyse long chain acids underivatised, because they are easy to derivatise and then they behave nicely in GC. For C2-C6 acids present in water, the derivatisation procedure is difficult, because extraction of acids from the matrix with organic solvents is non-quantitative. In this case, analysis of the free acids is possible, if extremely difficult. Rod has some nice posts previously on this issue. The first thing to do if you have to do this analysis is to inject the sample directly on the column, using a packed column, or a capillary on-column injector. These methods are fraught with difficulties as Rod has pointed out.

The "triangles" that Bruce refers to might be the result of the poor solubility of free fatty acids on phases designed for use with derivatised acids (usually non-polar or slightly polar phases), but I'm guessing the circumstances under which Bruce has observed this effect.

Rod-I'm sorry if the bit about your retirement is true. I have much enjoyed your posts. I hope you might continue to post sometimes, if only to correct any sophistry that contradicts your wealth of experience....

[sdegrace]

So... if, for the sake of argument, I was handed some forced degradation samples, say degraded with base, and the acid value was very high leading me to suspect by esters might be hydrolysed, I then have a pretty good idea where my peaks are, i.e., crudding up my liner and stuck on the front of my column .

[chromatographer1]

Yes, I believe your understanding is correct.

Rodney George

[sdegrace]

So hypothetically if I'm handed such a sample I'd better derivatize it lol.

[chromatographer1]

If I were the one who was required to do the maintenance on the GC and I only cared about HOW MUCH of the ester sample was degraded to the free acids and what was the present distribution of the esters remaining, I would send my sample through a SPE and separate the free acids from the esters. Then I would test the remaining ester sample and recover the free acids and esterify them to characterize their content. And I would have a much cleaner GC system and my column would last much longer.

But do what you are told to do.

Five more days until I retire and get to do whatever I DECIDE to do.

That will be an interesting change, no?

best wishes,

Rod the short timer

[DR]

[hijack]
Congrats, short timer! Enjoy your retirement.

Will we still see you in here from time to time?

[/hijack]

[skunked_once]

Rod,

Five more days until I retire and get to do whatever I DECIDE to do.

Are you not married then? Otherwise there would be an extensive honeydew list (Apologies in advance if this question is too personal.)

[chromatographer1]

I could not say what I said if I were married.

No other comment is required as all you married men know the other answer.

Must not forget to pack my telescope and my fishing pole. That should fill my schedule for nights and mornings. I guess I will have to nap in the afternoons.

I will be moving back to Missouri after a decade of living in beautiful central Pennsylvania, a lovely state with laws and taxes which are not to my tastes for retirement. But this Ozark farmboy will miss the lab and the joys of discovery. Only this year I discovered that the conventional wisdom, "CO2 does not elute from molecular sieve 5A as a peak" is inaccurate, as long as you use good 5A packed well. Another discovery was Neon and Helium can be separated by GC at room temperature. Little discoveries without economic benefit like these are still satisfying to a little mind filled with awe over the wonders of the universe.

I hope you all keep learning and are able to enjoy your trials in chromatography. (Hmm, what a title for a book or a scifi movie ? ! )

As Bruce says so often, "Keep having fun".

Just 4 more days to go.

Rod

[sdegrace]

Well Rod, I missed your replies because I was on vacation (far from any computer - my idea of a vacation!!), but if you're still reading the forum, happy retirement and a huge thanks for all the help you've generously given!!

[sdegrace]

Just to follow up, we thoroughly hydrolyzed a sample of fatty acid esters, and just for fun, I decided to inject them directly, then re-derivatize them and inject them like that. The result was exactly as expected - in the hydrolyzed sample consisting of FFAs, the fatty acids are essentially invisible using the particular method (Restek FAMEWAX, hydrogen carrier 20 cm/sec, oven gradient). The re-derivatized samples looked and assayed completely normal. So now we know for sure